{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/370287"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/370287","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Investigating the functional roles of the viral frameshift protein 2B* in EMCV infection","abstract":"Encephalomyocarditis virus (EMCV) is capable of infecting a wide range of mammalian hosts, causing encephalitis, diabetes, myocarditis in mice and primates as well as reproductive disorders in pigs. The severe pathogenicity is considered to be a result of the rapid cell lysis caused by EMCV. This highly efficient method of viral release also induces a severe inflammatory response, leading to disease and death. Viral propagation is further heightened by the ability of EMCV proteins to antagonise the host innate immune system, reducing the antiviral response. In 2011 the genome of EMCV was found to encode a thirteenth viral protein, 2B*, in an overlapping open reading frame within the gene for 2B. 2B* is translated via programmed ribosomal frameshifting (PRF). Unusually, the PRF is temporally regulated, increasing in efficiency from 0% at early stages of infection to 70% by 6-8 hours post-infection (hpi), thus resulting in time-dependent translation of 2B*. Truncation mutation of 2B* had previously been shown to result in a small plaque phenotype, indicative of a reduction in viral spread. However, the mechanism by which 2B* affects viral spread was left undetermined and the 2B* protein remained uncharacterised. The work presented here utilises time-course experiments to investigate the source of the small-plaque phenotype associated with loss of 2B*. Viral growth curves and cytotoxicity assays indicate that 2B* temporally regulates lytic virus release. A combination of flow cytometry, live-cell imaging and immunoblotting identified an increase in caspase-3 activation and gasdermin E (GSDME) cleavage in WT EMCV-infected samples relative to infection with 2B\\*KO EMCV prior to 24 hpi. Work with caspase KO cell lines confirmed that EMCV-induced rapid cell lysis is supported by caspase-3 but that WT EMCV can also induce a caspase-3-independent lysis pathway, which 2B\\*KO EMCV cannot. Together, these results show that timed production of 2B* enables temporal regulation of lytic virus release via both a caspase-3-mediated cell lysis pathway, likely to be GSDME-mediated pyroptosis, and a second caspase-3-independent pathway. The synergistic relationship between 2B* and caspase-3-mediated cell lysis means that EMCV can induce lysis independently of one of either caspase-3 or 2B* but when both are present lysis occurs more rapidly. Furthermore, interaction partners of 2B* were identified and characterised, both during infection and overexpression. A tagged virus allowed immunoprecipitation mass-spectrometry (IP-MS) of tagged-2B* and its interaction partners during infection, leading to the identification of an interaction between 2B* and every member of the 14-3-3 family of cellular proteins. As viral proteins which antagonise the innate immune response by sequestering 14-3-3 proteins had previously been identified for other virus species, this was investigated as a potential role of 2B*. Following identification of the 14-3-3 binding site of 2B* using bioinformatic prediction tools, we mutated this motif and this was shown to ablate the interaction. Further work confirmed that 2B* impedes innate immune signalling via this motif, with transcription of IFNB1 and IL6 being reduced following transient transfection. In summary, the findings of this thesis have uncovered two seemingly unrelated functions of 2B*: 1) promoting lytic virus release via caspase-3 activation in addition to a second caspase-3-independent pathway, and 2) antagonising innate immune signalling via interaction with the cellular 14-3-3 proteins. This study confirms that 2B* is a functional accessory protein and not a mere biproduct of a functionally important frameshift. Furthermore, this work contributes to the understanding of EMCV pathogenesis by uncovering novel virus-host interactions and identifying 2B* as the source of increased efficiency in EMCV-induced cell lysis relative to other picornaviruses.","abstract_html":"Encephalomyocarditis virus (EMCV) is capable of infecting a wide range of mammalian hosts, causing encephalitis, diabetes, myocarditis in mice and primates as well as reproductive disorders in pigs. The severe pathogenicity is considered to be a result of the rapid cell lysis caused by EMCV. This highly efficient method of viral release also induces a severe inflammatory response, leading to disease and death. Viral propagation is further heightened by the ability of EMCV proteins to antagonise the host innate immune system, reducing the antiviral response. In 2011 the genome of EMCV was found to encode a thirteenth viral protein, 2B*, in an overlapping open reading frame within the gene for 2B. 2B* is translated via programmed ribosomal frameshifting (PRF). Unusually, the PRF is temporally regulated, increasing in efficiency from 0% at early stages of infection to 70% by 6-8 hours post-infection (hpi), thus resulting in time-dependent translation of 2B*. Truncation mutation of 2B* had previously been shown to result in a small plaque phenotype, indicative of a reduction in viral spread. However, the mechanism by which 2B* affects viral spread was left undetermined and the 2B* protein remained uncharacterised. The work presented here utilises time-course experiments to investigate the source of the small-plaque phenotype associated with loss of 2B*. Viral growth curves and cytotoxicity assays indicate that 2B* temporally regulates lytic virus release. A combination of flow cytometry, live-cell imaging and immunoblotting identified an increase in caspase-3 activation and gasdermin E (GSDME) cleavage in WT EMCV-infected samples relative to infection with 2B\\*KO EMCV prior to 24 hpi. Work with caspase KO cell lines confirmed that EMCV-induced rapid cell lysis is supported by caspase-3 but that WT EMCV can also induce a caspase-3-independent lysis pathway, which 2B\\*KO EMCV cannot. Together, these results show that timed production of 2B* enables temporal regulation of lytic virus release via both a caspase-3-mediated cell lysis pathway, likely to be GSDME-mediated pyroptosis, and a second caspase-3-independent pathway. The synergistic relationship between 2B* and caspase-3-mediated cell lysis means that EMCV can induce lysis independently of one of either caspase-3 or 2B* but when both are present lysis occurs more rapidly. Furthermore, interaction partners of 2B* were identified and characterised, both during infection and overexpression. A tagged virus allowed immunoprecipitation mass-spectrometry (IP-MS) of tagged-2B* and its interaction partners during infection, leading to the identification of an interaction between 2B* and every member of the 14-3-3 family of cellular proteins. As viral proteins which antagonise the innate immune response by sequestering 14-3-3 proteins had previously been identified for other virus species, this was investigated as a potential role of 2B*. Following identification of the 14-3-3 binding site of 2B* using bioinformatic prediction tools, we mutated this motif and this was shown to ablate the interaction. Further work confirmed that 2B* impedes innate immune signalling via this motif, with transcription of IFNB1 and IL6 being reduced following transient transfection. In summary, the findings of this thesis have uncovered two seemingly unrelated functions of 2B*: 1) promoting lytic virus release via caspase-3 activation in addition to a second caspase-3-independent pathway, and 2) antagonising innate immune signalling via interaction with the cellular 14-3-3 proteins. This study confirms that 2B* is a functional accessory protein and not a mere biproduct of a functionally important frameshift. Furthermore, this work contributes to the understanding of EMCV pathogenesis by uncovering novel virus-host interactions and identifying 2B* as the source of increased efficiency in EMCV-induced cell lysis relative to other picornaviruses.","abstract_has_math":false,"creators":["Nguyen, Samantha"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Firth, Andrew","Stewart, Hazel"],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024-03-28","date_published":"2024-03-28","updated_at":"2026-07-22T22:23:57Z","subjects":["14-3-3","caspase","EMCV","innate immune antagonism","Lytic release","Picornavirus"],"languages":["eng"],"rights":[],"rights_urls":["https://www.repository.cam.ac.uk/bitstreams/c0c7ffa2-0bdb-45b3-9368-4358ef32cea7/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.109746","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Firth, Andrew","Stewart, Hazel"]},{"key":"dc:creator","label":"Author","values":["Nguyen, Samantha"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2024-03-28"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/370287"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["14-3-3","caspase","EMCV","innate immune antagonism","Lytic release","Picornavirus"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.repository.cam.ac.uk/bitstreams/c0c7ffa2-0bdb-45b3-9368-4358ef32cea7/download","https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.109746"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/ac2a49bc-ea7f-4355-b1e8-83c6be39e3cb/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Encephalomyocarditis virus (EMCV) is capable of infecting a wide range of mammalian hosts, causing encephalitis, diabetes, myocarditis in mice and primates as well as reproductive disorders in pigs. The severe pathogenicity is considered to be a result of the rapid cell lysis caused by EMCV. This highly efficient method of viral release also induces a severe inflammatory response, leading to disease and death. Viral propagation is further heightened by the ability of EMCV proteins to antagonise the host innate immune system, reducing the antiviral response. In 2011 the genome of EMCV was found to encode a thirteenth viral protein, 2B*, in an overlapping open reading frame within the gene for 2B. 2B* is translated via programmed ribosomal frameshifting (PRF). Unusually, the PRF is temporally regulated, increasing in efficiency from 0% at early stages of infection to 70% by 6-8 hours post-infection (hpi), thus resulting in time-dependent translation of 2B*. Truncation mutation of 2B* had previously been shown to result in a small plaque phenotype, indicative of a reduction in viral spread. However, the mechanism by which 2B* affects viral spread was left undetermined and the 2B* protein remained uncharacterised. The work presented here utilises time-course experiments to investigate the source of the small-plaque phenotype associated with loss of 2B*. Viral growth curves and cytotoxicity assays indicate that 2B* temporally regulates lytic virus release. A combination of flow cytometry, live-cell imaging and immunoblotting identified an increase in caspase-3 activation and gasdermin E (GSDME) cleavage in WT EMCV-infected samples relative to infection with 2B\\*KO EMCV prior to 24 hpi. Work with caspase KO cell lines confirmed that EMCV-induced rapid cell lysis is supported by caspase-3 but that WT EMCV can also induce a caspase-3-independent lysis pathway, which 2B\\*KO EMCV cannot. Together, these results show that timed production of 2B* enables temporal regulation of lytic virus release via both a caspase-3-mediated cell lysis pathway, likely to be GSDME-mediated pyroptosis, and a second caspase-3-independent pathway. The synergistic relationship between 2B* and caspase-3-mediated cell lysis means that EMCV can induce lysis independently of one of either caspase-3 or 2B* but when both are present lysis occurs more rapidly. Furthermore, interaction partners of 2B* were identified and characterised, both during infection and overexpression. A tagged virus allowed immunoprecipitation mass-spectrometry (IP-MS) of tagged-2B* and its interaction partners during infection, leading to the identification of an interaction between 2B* and every member of the 14-3-3 family of cellular proteins. As viral proteins which antagonise the innate immune response by sequestering 14-3-3 proteins had previously been identified for other virus species, this was investigated as a potential role of 2B*. Following identification of the 14-3-3 binding site of 2B* using bioinformatic prediction tools, we mutated this motif and this was shown to ablate the interaction. Further work confirmed that 2B* impedes innate immune signalling via this motif, with transcription of IFNB1 and IL6 being reduced following transient transfection. In summary, the findings of this thesis have uncovered two seemingly unrelated functions of 2B*: 1) promoting lytic virus release via caspase-3 activation in addition to a second caspase-3-independent pathway, and 2) antagonising innate immune signalling via interaction with the cellular 14-3-3 proteins. This study confirms that 2B* is a functional accessory protein and not a mere biproduct of a functionally important frameshift. 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This highly efficient method of viral release also induces a severe inflammatory response, leading to disease and death. Viral propagation is further heightened by the ability of EMCV proteins to antagonise the host innate immune system, reducing the antiviral response. In 2011 the genome of EMCV was found to encode a thirteenth viral protein, 2B*, in an overlapping open reading frame within the gene for 2B. 2B* is translated via programmed ribosomal frameshifting (PRF). Unusually, the PRF is temporally regulated, increasing in efficiency from 0% at early stages of infection to 70% by 6-8 hours post-infection (hpi), thus resulting in time-dependent translation of 2B*. Truncation mutation of 2B* had previously been shown to result in a small plaque phenotype, indicative of a reduction in viral spread. However, the mechanism by which 2B* affects viral spread was left undetermined and the 2B* protein remained uncharacterised. The work presented here utilises time-course experiments to investigate the source of the small-plaque phenotype associated with loss of 2B*. Viral growth curves and cytotoxicity assays indicate that 2B* temporally regulates lytic virus release. A combination of flow cytometry, live-cell imaging and immunoblotting identified an increase in caspase-3 activation and gasdermin E (GSDME) cleavage in WT EMCV-infected samples relative to infection with 2B\\*KO EMCV prior to 24 hpi. Work with caspase KO cell lines confirmed that EMCV-induced rapid cell lysis is supported by caspase-3 but that WT EMCV can also induce a caspase-3-independent lysis pathway, which 2B\\*KO EMCV cannot. Together, these results show that timed production of 2B* enables temporal regulation of lytic virus release via both a caspase-3-mediated cell lysis pathway, likely to be GSDME-mediated pyroptosis, and a second caspase-3-independent pathway. The synergistic relationship between 2B* and caspase-3-mediated cell lysis means that EMCV can induce lysis independently of one of either caspase-3 or 2B* but when both are present lysis occurs more rapidly. Furthermore, interaction partners of 2B* were identified and characterised, both during infection and overexpression. A tagged virus allowed immunoprecipitation mass-spectrometry (IP-MS) of tagged-2B* and its interaction partners during infection, leading to the identification of an interaction between 2B* and every member of the 14-3-3 family of cellular proteins. As viral proteins which antagonise the innate immune response by sequestering 14-3-3 proteins had previously been identified for other virus species, this was investigated as a potential role of 2B*. Following identification of the 14-3-3 binding site of 2B* using bioinformatic prediction tools, we mutated this motif and this was shown to ablate the interaction. Further work confirmed that 2B* impedes innate immune signalling via this motif, with transcription of IFNB1 and IL6 being reduced following transient transfection. In summary, the findings of this thesis have uncovered two seemingly unrelated functions of 2B*: 1) promoting lytic virus release via caspase-3 activation in addition to a second caspase-3-independent pathway, and 2) antagonising innate immune signalling via interaction with the cellular 14-3-3 proteins. This study confirms that 2B* is a functional accessory protein and not a mere biproduct of a functionally important frameshift. 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